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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2010, April 11 to 2010, May 30
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP with certificate. The study is clear and complete in any parts. The substance is well identified, the method is suitable for the substance. Used for read across
Reason / purpose:
reference to same study

NOAEL subcronic toxicity rat = 1000 mg/kg bw/day

Toxic effect type:
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
1 000 mg/kg bw/day
Study duration:

A sub-chronic toxicity study (90 days) is not proposed by the registrant because the frequency and duration of human exposure indicates that a longer term study is not appropriate; the exposure to the substance is likely in the working scenario, where exposure of worker to biodiesel is pertinent only for accidental dermal route, and RMM are already applied. Inhalation route is not considered due to the physical chemical properties of the substance.

Furthermore the substance don't have dangerous properties that cannot be detected in a short-term toxicity study; accumulation of the substance or its metabolites in certain tissues or organs which would possibly remain undetected in a shortterm

toxicity study don't are liable to result in adverse effects after prolonged exposure. They enter in the endogen metabolism of fatty acids and fatty acid methyl esters that can be accumulated or released by tissues on demand.

A NOAEL can be established because of the absence of adverse effect both in 28 and 90 days. Route to route estrapolation can be done from oral to dermal.

Classification for STOT is relevant where significant toxic effects are seen from single or repeated dose studies. No such significant toxicity is seen in all available studies

No classification for STOT is warrented at present under 67/548/EEC or Regulation 1272/2008.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
- Name of test material : Metiloil A
- Physical state: yellow liquid
- Storage condition of test material: at room temperature
- Composition of test material, percentage of components: mixture of methylesthers of satured and insatured C16to 18 fatty acids, no data
- Lot/batch No.: 1169
- Acid number: 7 mgKOH/g
Specific details on test material used for the study:
Test Item Name: C16-C18 and C18 Unsaturated Alkyl Methyl EsterBatch/Lot No: 110-1ECAS Number: 67762-38-3
Description: Yellow Liquid
Expiry / Retest: A sample of the test item was sent to ASG Analytik Service forester content (stability testing) following the end of the dosingperiod.
Purity: 99.35%Storage Conditions: 2-8°C protected from light

Test animals

Details on test animals and environmental conditions:
One batch of 42 male and 42 female Sprague-Dawley rats (Crl: CD®(SD) strain) were received on 30 March 2010 from Charles River UK Limited, Margate, Kent, UK. The animals were ca 6 weeks of age and weighed 201-239 g for males and 150-179 g for females, on despatch.

Each animal received a subcutaneously implanted electronic chip, which identified it individually within the study, and which corresponded to that animal’s number. Each animal was given a cage card which was colour coded for treatment group, and was marked with the study number, cage and animal numbers, sex and relevant treatment group. In addition, a second cage card was given to each animal identifying that animal by study
number, neurotox identification, animal number and sex. This cage card was not colour coded.

The animals were acclimatised in the Charles River animal room for 12 days prior to commencement of treatment. All the animals were examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on the study.

There was automatic control of light cycle, temperature and humidity in the animal room. Light hours were 0700-1900 h. The target ranges for temperature and humidity were 21°C ± 2°C and 55% ± 15% respectively, with a minimum of 15 air changes per hour.
Daily monitoring indicated that temperature remained within the target range of 21°C ± 2°C (actual range 20°C-23°C) and humidity was slightly above the target range on 6 occasions (actual range 35-68%). This deviation from target range was considered to be minor and had no significant impact on the outcome and integrity of the study.

Each day, floors were swept and then mopped with a 0.5% solution of Tego 2000 (Th.Goldschmidt Limited, Ruislip, Middlesex, UK), an amphoteric biocide/cleanser. The room was washed with this solution at approximately weekly intervals.

The animals were initially housed 2 per cage, in polycarbonate cages, with solid bottoms and stainless steel mesh tops and measured ca 48 x 37.5 x 25 cm. A stainless steel food hopper and a polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual cages of a
stainless steel grid insert measuring ca 48 x 37.5 x 25 cm. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. The mated females were transferred to individual solid bottomed cages measuring ca 48 x 37.5 x 25 cm. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters retained this cage type until termination. After mating the males remained singly housed until termination.

Cages, absorbent papers and water bottles were changed at regular intervals, as appropriate. White tissue paper nesting material was changed when it was unacceptably soiled. Clean wood shavings were provided at each change of solid-bottomed cage.

To provide environmental enrichment, wooden chewsticks were made available to the animals as appropriate.

Rat and Mouse Breeder Diet No. 3 (Expanded) SQC supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used in this study is retained in the study archive.
Due to technical error, Animal 74 (Group 4 female) was fed expired diet from 02-07 June 2010 prior to terminal kill. The diet given expired on the 01 June 2010; therefore this deviation was considered to be minor given that the food had expired on the previous day to use and the short length of time the animal had consumed the expired diet. This protocol deviation had no impact on the outcome or integrity of the study.
The food was not considered to contain any additional substances in sufficient concentration to influence the outcome of the study.
The animals had access to domestic, mains quality water ad libitum. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.

Cages were allocated to treatment group by the use of randomly sequenced numbers, in such a way that each complete rack contained representatives from all treatment groups.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Dose levels were agreed upon with the Sponsor after evaluation of existing relevant toxicological data, including Charles River Study 495283; a one week dose range finding study in rats. Results from this study indicated no adverse effect of treatment at 2000 mg/kg/day. However, for the purposes of this study a high dose level of 1000 mg/kg/day was considered appropriate.

The animals were dosed once daily by oral gavage at a dose volume of 5 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined on each day by the weight of the animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until parturition was complete, the dose volume of the females was determined by the weight of the animal on Day 16 of gestation.
The males were dosed once daily for 4 weeks overall, commencing 2 weeks prior to mating. The females were dosed once daily from 2 weeks prior to mating then continued until at least Day 4 of lactation. Dosing for males and females continued until the day prior to termination.
Details on mating procedure:
A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages.
Pairing was on a 1 male to 1 female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected or 14 nights had elapsed.
Vaginal lavages were taken daily early each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:
Duration of treatment / exposure:
4 weeks for males,
2 weeks before pairing, during pairing, during gestation and until 4 days post partum for female
Frequency of treatment:
Once daily
Details on study schedule:
The females were allowed to litter normally. The day of birth of the litter was designated Day 0 of lactation. The duration of gestation in days was calculated and evaluated. The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up
to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Pups killed on Day 7 of lactation were also weighed en masse by sex; this additional data has not been reported and will be retained in the raw data. This protocol deviation had no significant impact on the outcome or integrity of the study.
When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any externally normal decedent pups were discarded. Any deficiencies in maternal care were recorded. Points looked for were inadequate construction and cleaning of the nest, pups left scattered and cold, physical harm of pups, or apparently inadequate lactation or feeding. Detailed information is retained in the study data.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0 mg/kg/day
actual ingested
Doses / Concentrations:
100 mg/kg/day
actual ingested
Doses / Concentrations:
300 mg/kg/day
actual ingested
Doses / Concentrations:
1000 mg/kg/day
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle


Parental animals: Observations and examinations:
All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination to include body orifices (ears, nostrils, mouth, anus, vulva) and cranial, thoracic and abdominal organs and tissues. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number.

Adults were examinated for the followings (for more details, see the related ) Repeated doese toxicity study):
Mortality check
Clinical observation
Body weight
Food consumption
Water consumption
Ophthalmic examinations
Functional observation
Function test
Clinical chemistry
Bone marrow smears

All observation didn't reveal any effect related to the treatment
Litter observations:
Mating performance or fertility (as measured by the fertility indices) was not affected by
There were no obvious effects on the duration of gestation at any dose level applied.
There were no effects on the number of live young born at any of the dose levels, or on the
mean number of implant sites per pregnancy.

The following abnormalities were observed among pups:
Litter 45 (Group 1): Day 1-2: One female pup with no tail. Pup was found dead on Day 2.
The following observations were observed among pups:
The following is a summary of the observations recorded among pups. Full details of the
observations noted within the litters are retained within the raw data.
Litter 41 (Group 1): Day 1: One female pup with bruising to the head.
Litter 47 (Group 1): Day 1-2: One female pup scabbing on head
Litter 48 (Group 1): Day 3: One male pup scabbing present on muzzle
Litter 58 (Group 2): Day 1-2: One male pup scabbing on anus.
Litter 58 (Group 2): Day 1: One male and one female pup bruising on dorsal surface.
Litter 61 (Group 3): Day 1: One male pup had bruising on muzzle
Litter 74 (Group 4): Day 1-2: One male pup with bruising to the back.
Litter 74 (Group 4): Day 3-7: One male pup with scabbing to the muzzle
Litter 79 (Group 4): Day 2-3: One male pup with bruising to the dorsal surface.
These observations were considered to be typical for pre-weanlings.

Litter Survival, Litter and Pup Weights:
At 1000 mg/kg/day the survival of pups (as indicated by the viability index) was slightly
lower (88%) compared to Control. This was due to Animal 72 which had a total litter loss;
this incidence and percentage are well within the background data for this laboratory.
Mean litter and pup weights in treated groups were similar to Control.
Postmortem examinations (parental animals):
See repeated toxicity study results
Postmortem examinations (offspring):
Offspring killed or found dead were sexed, then checked for the presence of milk in the stomach and the presence of externally visible abnormalities. Any abnormal pups were preserved in 10% formalin or methylated ethyl alcohol as appropriate for possible future examination. Externally normal decedents were discarded.
Reproductive indices:
The following indices of fertility were evaluated:

For each group:
Fertility Index (male) = Number siring a litter/Number paired
Fertility Index (female) = Number pregnant/Number paired
Gestation Index = Number bearing live pups/Number pregnant

For each litter and group:
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth Index =Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: none adverse effect

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
1000 mg/kg/day
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: none adverse effect

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

The tested substance revealed no effect in combined Repeated Dose Toxicity Study with the reproduction/Developmental Toxicity screening Test in Ratsfor a dose of until 1000 mg/kg/bw
Executive summary:

A total of 80 rats (40 males and 40 females) Sprague-Dawley rats,Crl CD® (SD)were allocated to four groups, three of which received the test item, Heptadecanoic Fatty Acid Methyl Ester (C16-C18) while the other group received the vehicle (corn oil) by oral route (gavage) once daily under a dosage volume of 5 mL/kg. Animals will be dosed for 2 weeks prior to pairing, during pairing, during gestation and until at least day 4 post-partum for females or until sacrifice for non pregnant females and until necropsy for males following 4 weeks of treatment.

The following dose-levels were used:

. Group 1 (10 males and 10 females): 0 mg/kg/day,

. Group 2 (10 males and 10 females):100mg/kg/day,

. Group 3 (10 males and 10 females):300mg/kg/day,

. Group 4 (10 males and 10 females):1000mg/kg/day.